This research will be done primarily in Russia at Moscow State University in collaboration with Dr. William Merrick as an extension of the research being conducted in his laboratory. The project is concerned with studies of cellular internal ribosome entry sites (IRESs) of eukaryotic cells. The cellular IRESs direct mRNA translation using atypical mechanisms of recognition of the initiation start codon. They are involved in many important cellular processes and responses: genome evolution, cell-division, apoptosis, cell differentiation, response to nutritional stimuli etc. However, unlike some of the viral IRESs, for none of the cellular IRESs is the mechanism of functioning known. Their secondary structures do not resemble those of known viral IRESs and are dissimilar among themselves. The proposal aims to dissect molecular mechanisms of translation initiation for some of cellular IRESs mostly using a powerful modern technique of assembly of translation initiation complexes from purified components combined with the toeprint assay. For this study, three cellular IRESs were selected on the base of their efficient translation in a cell-free system and therefore presumed simpler requirement for translation initiation components to perform their function: the IRES of the mRNA encoding NRF (NF-Kb repressing factor), the intercistronic IRES from a natural dicistronic mRNA transcribed from human retrotransposon L1, and that directing translation of human Hsp 70 mRNA. The translation initiation complexes on these IRESs will be assembled using purified translational components and the initiation factor requirements will be determined. The boundaries of these IRESs and the most important functional sites will be defined by deletion analysis. Each of the mutants produced will be tested for its ability to form translation initiation complexes and to maintain the IRES-activity in transfected cells. The translational components that bind to the critical functional sites of the IRESs will be determined by footprinting.